Antiviral screening assays

Four basic approaches are conducted for plant selection for antiviral screening assays 1- Random collection of plants followed by mass screening. 2- Ethnomedical approach. 3- Literature-based follow up of the existing leads. 4- Chemotaxonomic approach [12]. The second and third approaches are the most favored ones because of their cost-effective applicability. The selection based on folkloric use proved five times higher percentage of active leads than other approaches. The random approach usually affords more novel compounds with antiviral activity. Combining ethnomedical, phytochemical and taxonomical approaches is considered the best compromise. 

The literature on antiviral screening for HHV-6 and HHV-7 is sparse and has been reviewed elsewhere (1,3-5). Cell systems for viral propagation have included primary human peripheral blood and cord blood lymphocytes (CBL), and T-cell lines. Methods for monitoring viral replication have included examining infected cultures for cytopathic effect, immunofluorescence analysis, quantitation of viral DNA, and focus-forming assays. Phosphonoformic acid (foscarnet), phosphonoacetic acid, and ganciclovir inhibit HHV-6 and HHV-7... 

Ender A et al. (2004) Screening of blood donations for HIV-1 and HCV RNA by transcription-mediated amplification assay one year experience. Transfus Med Hemother. 31 10-15. Kasermann F, Kempf C (1997) Photodynamic inactivation of enveloped viruses by buckminster-fullerene. Antiviral Res. 34 65-70. 

As a conclusion, these findings provide additional evidence for the supposition that the assays mentioned above play the part of useful primary screening in the survey of bioactive natural products. For the reasons outlined above, it is very important to focus on plants to discern novel antivi-ral/antimicrobial compounds. Therefore, we truly hope that our studies, as well as similar reports by different researchers, may help find new antimicro-bials/antivirals from herbal sources. 

Weislow, O. S., Kiser, R., Fine, D. L., Bader, J., Shoemaker, R. H. and Boyd, M. R. (1989). New soluble-formazan assay for HIV-1 cytopathic effects application to high-flux screening of synthetic and natural products for AIDS-antiviral activity J. Natl. Cancer Inst. 81, 577-586 (published erratum appears in J. Natl. Cancer Inst. 81, 963). 

In order to find new sources of antiviral agents with different mechanisms of action, extracts of marine algae from all over the world were assayed for anti-HSV activity. The first screening of 89 types of seaweed collected from British Columbia, Canada and Korea for antiviral activity was reported by Kim et al. [66]. Analipus japonicus was the most potent anti-herpes algae. Extracts from 13 types of Korean seaweed previously shown to contain antiviral activity were investigated in more detail in order to learn their mechanism of action [14]. Four species, Enteromorpha linza, Colpomenia bullosa, Scytosiphon lomentaria and Undaria pinnatifida were active against HSV. In experiments to determine the site of action of these antiviral extracts, the predominant activity was virucidal (i.e., direct inactivation of virus particles) rather than inhibition of virus replication. 

Advances in the screening of chemical libraries has led to the discovery of nonnucleoside inhibitors of RT (NNRTIs). Two approaches can be followed. When an appropriate antiviral assay is available compounds can be screened in whole cells. These assays are frequently labor intensive and therefore generally have a lower throughput than isolated enzyme assays. These assays have the advantage that chemical leads generated by this approach have already overcome the barriers of cell penetration and stability. 

The discovery of another NNRTI, nevirapine, illustrates the second approach (see Scheme 6). Here it is no longer necessary to have access to an antiviral assay to discover active entities. Compounds are screened against an isolated enzyme. High throughput of compounds in the screen is a key advantage of this method enabling the testing of thousands of compounds however, com-... 

This assay format is well suited to the screening of test compounds for potential antiviral activity. The assay provides a minimal assessment of antiviral activity by measuring the levels of HBV virion release from the cells, as well as providing a measurement of cytotoxicity (see Subheading 3.2.2.), Two rows of cells will be required for each compound, plus four rows for the assay controls (two for untreated, and two for positive antiviral control (e.g., 3TC). After treating for 9 d, the media are harvested from the antiviral plates and transferred to 96-well U-bottomed plates. They are then centrifuged, and supernatant is transferred to tubes for dot-blot hybridization analysis of HBV virion DNA. The medium is aspirated off of the toxicity plates and discarded. Toxicity plates are then incubated with neutral red dye (methylthiouracil [MTT] can also be used if preferred), washed with DPBS, developed with an acetic acid/ ethanol solution, and assayed in a plate reader. 

This chapter will describe systems for culturing HHV-6 and HHV-7 as well as assays for their replication that can be used to screen potential antiviral compounds. 

Some compounds may not actually reduce the amount of p24 released from the cell but cause the release of noninfectious virus particles. Such examples are the glycosidase inhibitors. In this assay, supernatants from the primary screen containing different concentrations of the drug are added to indicator cells and the extent of the CPE produced in the indicator cells gives a measure of the antiviral activity. C8166 and CEM cells form syncytia readily but MT2, MT4, and JM cells form syncytia that are more discrete and easier to count. 

A sensitive method to measure toxic effects of drugs is to monitor cellular metabolism by incorporation of 3H-thymidine or 14C-protein hydrolysate. 14C-protein is used in preference to 3H-thymidine when it is necessary to avoid competition in uptake between the labeled thymidine and an unlabeled nucleoside reverse-transcriptase inhibitor. These methods are used mainly to monitor sublethal toxicity after initial screening or when comparing structure-activity relationships. The assays described here are carried out using 6-mL culture tubes (Note 1). These assays are carried out in parallel with the antiviral assay. 

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